CD105+CD90+CD13+ identifies a clonogenic subset of adventitial lung fibroblasts

Mesenchymal cells are important components of specified niches in the lung, and can mediate a wide range of processes including tissue regeneration and repair. Dysregulation of these processes can lead to improper remodeling of tissue as observed in several lung diseases. The mesenchymal cells responsible remain poorly described, partially due to the heterogenic nature of the mesenchymal compartment and the absence of appropriate markers. Here, we describe that CD105+CD90+ mesenchymal cells can be divided into two populations based on their expression of CD13/aminopeptidase N (CD105+CD90+CD13− and CD105+CD90+CD13+). By prospective isolation using FACS, we show that both these populations give rise to clonogenic fibroblast-like cells, but with an increased clonogenic and proliferative capacity of CD105+CD90+CD13+ cells. Transcriptomic and spatial analysis pinpoints an adventitial fibroblast subset as the origin of CD105+CD90+CD13+ clonogenic mesenchymal cells in human lung.


Sample preparation for data-independent acquisition (DIA) mass spectrometry
Cell layers from cultured mesenchymal cells were harvested with 10 mM Hepes buffer (pH 8) containing 10 mM DTT and 4% SDS using a cell scraper. Collected cell layers were heated at 95°C for 5 minutes followed by sonication using a Bioruptor Plus (Diagenode) for 10 minutes with cycles of 15 seconds on and 15 seconds off at high power. Cell lysates were alkylated with 50 mM iodoacetamide (IAA, Sigma) for 45 minutes at room temperature. To generate tryptic peptides and remove contaminants Single-Pot Solid-Phase-enhanced Sample Preparation (SP3) was performed utilizing paramagnetic beads (Thermo Fisher Sera-Mag Speed Beads A and B; Sigma-Aldrich) 4 . Briefly, protein samples were first dried and resuspended in liquid chromatography-grade water, then incubated with beads in 50% acetonitrile (ACN) containing 0.17% formic acid (FA) for 10 minutes to bind proteins to beads. Placed on a magnet, beadbound proteins were washed with 70% ethanol and then with 100% ACN. Off the magnet, tryptic digestion was performed on the immobilized proteins by incubating in 100 mM ammonium bicarbonate (Sigma-Aldrich) containing 250 ng/µL trypsin (sequencing grade modified trypsin porcine; Promega, Madison, USA) overnight (16 h) at 37°C. The peptide-bead mixture was then diluted with ACN to 95% ACN and incubated for 10 minutes. Placed on the magnet again, the bead-bound peptides were washed with 100% ACN. Off the magnet, the peptides were eluted in 2% DMSO in water and mixed 1:1 (v/v) with a buffer containing 4% ACN, 0.4% FA and iRT peptides (1:10 v/v; Biognosys, Schlieren, Switzerland).

LC-MS/MS analysis
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed on a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific). Peptide separation was carried out by an EASY-nLC 1000 liquid chromatography system (Thermo Fisher Scientific) connected to an RP-HPLC EasySpray column (ID 75 µm x 25 cm C18 2 µm 100 Å resin; Thermo Fisher Scientific). For data-independent acquisition (DIA), a 120-minute linear gradient was run from 5% to 35% ACN, followed by 5 minutes from 35% to 95% ACN and 10 minutes at 95% ACN, using solvent A (2% ACN, 0.1 % FA) and solvent B (100% ACN, 0.1% FA) at a 300 nl/min flow rate. For DDA, a full MS scan at mass range 400-1600 m/z (resolution 70,000 at 200 m/z) was followed by MS/MS scans (resolution 17,500 at 200 m/z) of the top 15 most intense precursor ions fragmented by higher energy collision induced dissociation (HCD).
To trigger MS/MS scan of precursor ions a MS precursor intensity threshold was set to 1.7e4.

Mass spectrometry data analysis
Raw files from DDA and DIA analysis were converted to mzML files using MSconvert. All data analysis and searches were processed through openBIS 5 . X!Tandem was used to search data against the human UniProt FASTA database (version November 2015) with reversed decoy sequences. Fixed modification of cysteine carbamidomethylation and variable modifications of methionine oxidation and proline hydroxylation were included in the searches.
Mass tolerance for precursor ions and fragment ions was set to 20 ppm and 50 ppm, respectively. The Generated files were subsequently analyzed using peptideProphet, iProphet and MAYU in the Trans-Proteomic Pipeline (TPP, version 4.7) 6-8 . For quantification of DIA data a spectral library was created based on DDA data through workflows included in openBIS 9 , applying spectraST to generate target assays, CLI to calculate 1% FDR for peptides and proteins and TRIC to perform feature alignment 10 . Then, openSWATH was used to analyze DIA data 11 .
Using R (version 3.6.1) and R Studio software (version 1.2.1335), prototypic peptides were selected and analyzed using the DEqMS package to identify differentially expressed proteins (adjusted for donor ID and tissue localization) and to generate the volcano plot 12

Supplementary Tables and Figures
Supplementary